Project Summary Bacterial drug resistance is a worldwide problem that limits the effectiveness of antibiotics in the clinic. While there are several molecular mechanisms that contribute to drug resistant phenotypes, it is well established that efflux pumps play a prominent role in pathogenic bacteria. Indeed, multidrug transporters constitute a fundamental mechanism used by bacteria to survive in the presence of toxic compounds by binding and transporting a broad array of structurally diverse compounds. The long-term goals of this project are to discover novel mechanisms used by multidrug transporters and to harness this knowledge to predict and control function. In this competitive renewal, we are now poised to tackle the major challenge in the field of understanding how efflux pumps achieve broad drug specificity required for conferring multidrug resistance. To accomplish this goal, we need to establish a comprehensive understanding of the catalytic cycle for an efflux pump system amenable to detailed biological, biochemical and biophysical studies. For this reason, our proposal will use EmrE from the SMR family as the model drug transporter since it embodies the minimal level of complexity while retaining the key features shared among all secondary active efflux pumps. Aim 1 will test an occluded-state theory that we hypothesize is widely used by efflux pumps for drug binding. Aim 2 will seek to define the molecular basis for substrate-induced activation of dynamics versus inhibitor-induced repression of dynamics, as well as development of a computational platform for predicting binding and transport. Finally, Aim 3 will set out to determine the molecular basis of binding specificity versus promiscuity through a comparative analysis of two subfamilies within the SMR family that have markedly different specificity profiles. Each of these Aims works synergistically toward our long-term goal of articulating novel transport mechanisms and applying our knowledge to develop models for making predictions about function. A major strength of this project is the integrated nature of the approach which utilizes significant collaboration and a combination of biological, biophysical, and computational methods aimed at unveiling general transport mechanisms designed by nature and shared among other multidrug efflux pumps. The outcomes of this research will make a significant impact in understanding efflux-mediated multidrug resistance, and the approaches and methods developed will be translatable to knowledge discovery in other efflux systems.